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. 2010 Sep;6(9):3388-94.
doi: 10.1016/j.actbio.2010.03.025. Epub 2010 Mar 23.

Molecular resurfacing of cartilage with proteoglycan 4

K Chawla  1 , H O Ham, T Nguyen, P B Messersmith
Affiliations

Molecular resurfacing of cartilage with proteoglycan 4

K Chawla et al. Acta Biomater. 2010 Sep.

Abstract

Early loss of proteoglycan 4 (PRG4), a lubricating glycoprotein implicated in boundary lubrication, from the cartilage surface has been associated with degeneration of cartilage and early onset of osteoarthritis. Viscosupplementation with hyaluronic acid and other macromolecules has been proposed as a treatment of osteoarthritis. However, the efficacy of viscosupplementation is variable and may be influenced by the short residence time of lubricant in the knee joint after injection. Recent studies have demonstrated the use of aldehyde (CHO) modified extracellular matrix proteins for targeted adherence to a biological tissue surface. It is hypothesized that CHO could be exploited to enhance the binding of lubricating proteoglycans to the surface of PRG4-depleted cartilage. The objective of this study was to determine the feasibility of molecular resurfacing of cartilage with CHO-modified PRG4. PRG4 was chemically functionalized with aldehyde (PRG4-CHO) and aldehyde plus Oregon Green (OG) fluorophore (PRG4-OG-CHO) to allow for differentiation of endogenous and exogenous PRG4. Cartilage disks depleted of native PRG4 were then treated with solutions of PRG4, PRG4-CHO, or PRG4-OG-CHO and then assayed for the presence of PRG4 by immunohistochemistry, ELISA, and fluorescence imaging. Repletion of cartilage surfaces was significantly enhanced with the inclusion of CHO compared with repletion with unmodified PRG4. These findings suggest a generalized approach which may be used for molecular resurfacing of tissue surfaces with PRG4 and other lubricating biomolecules, perhaps leading in the future to a convenient method for overcoming loss of lubrication during the early stages of osteoarthritis.

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Figures

Figure 1
Figure 1
Chemical modification of PRG4 with Oregon Green (OG) and aldehyde (CHO). Purified PRG4 was reacted with succinimidyl ester of Oregon Green 488 carboxylic acid, or succinimidyl-4-formylbenzamide (S-4FB), generating PRG4-OG or PRG4-CHO, respectively. A portion of PRG4-OG was further reacted with S-4FB to generate PRG4 conjugated with both OG and CHO (PRG4-OG-CHO).
Figure 2
Figure 2
Schematic of methods used to investigate surface modification of cartilage disks with chemically modified PRG4.
Figure 3
Figure 3
Characterization of PRG4. (A) Western blot of purified PRG4 probed with mAb 4D6 after separation on a 4–15% gradient polyacrylamide gel. A band was visualized at ~345 kDa. (B) PRG4 (non-functionalized), PRG4-CHO, and PRG4-OG-CHO were visualized by Ruby Red fluorescent stain gel at ~345 kDa.
Figure 4
Figure 4
PRG4 content of native cartilage (white), cartilage depleted of native PRG4 by SDS treatment (gray), and cartilage repleted with PRG4 (striped), PRG4-CHO squares), and PRG4-OG-CHO (black). Mean±SEM, n= 4–6, (▲) p<0.001, (●) p<0.01, (◆) p<0.05.
Figure 5
Figure 5
Immunohistochemical localization of PRG4 at the articular surface in (A-i:E-i) no reaction (control), (A-ii:E-ii) SDS dissociated, or (A-iii:E-iii) PRG4, (A-iv:E-iv) PRG4-CHO, or (A-v:E-v) PRG4-OG-CHO repleted samples. Cartilage sections were probed with (C-i:E-v) mAb 4D6 or (A-i:B-v) non-specific mouse IgG. Images were grouped as (i) no reaction, (ii) SDS dissociated, or (iii) PRG4, (iv) PRG4-CHO, (v) PRG4-OG-CHO repleted samples. The letters (A–E) were used to denote the imaging field of view, i.e., IgG phase, IgG rhodamine, PRG4 phase, PRG4 OG, PRG4 rhodamine, for the aforementioned groupings. Bar = 0 μm.”
Figure 6
Figure 6
Potential mechanism of PRG4-CHO modification of the articular cartilage surface (not drawn to scale). A. PRG4-CHO conjugates react via Schiff base formation with primary amines present in ECM components of the cartilage surface. B. PRG4-CHO may bind to the tissue surface as monomers (i) or as multimeric complexes formed in solution by intermolecular Schiff base formation (ii, iii).
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